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Escherichia coli: the Lac operon
Lac operon is a group of genes encoding enzymes for lactose utilization in Escherichia coli. The operon consists of lacZ, which encodes β-glycosidase; lacY, which encodes permease; and lacA, which encodes galactoside transacetylase 1. The regulator of the operon, the Lac repressor, is encoded by an upstream gene lacI. lacZ, lacY and lacA are transcribed by a single promoter, producing a single mRNA; whereas lacI is transcribed by its own promoter 2. Permease is a membrane transporter that facilitates the uptake of lactose and β-glycosidase degrades lactose into galactose and glucose 2. The degradation of lactose can be visualized by degradation of artificial substrate (X-gal), production of acidic compounds (MacConkey agar) or reducing compounds (tetrazolium agar) 1. Since there are multiple easy ways to visualize the Lac phenotype, lac operon, especially lacZ is broadly used as reporter constructs. In the following, there are two examples of application of Lac operon in genetic analysis study. Transcriptional fusion -- distinguishing a repressor from an activator Transcription fusion means to put the promoterless lac gene under the control of a different promoter surrounding the gene of interest(your favorite gene, yfg). Thus the transcription level of yfg is the same as the reporter gene. Therefore the transcription level of yfg is able to be identified by measuring the transcription level of the reporter gene 1. An important application of transcriptional fusion is to distinguish a repressor from an activator 1. In a constitutive mutation – where the gene of interest is constitutively expressed, we can introduce a wild-type gene of the regulator in trans and perform a diploid analysis. If the transcription of the reporter gene (and the gene of interest) is significantly decreased, the regulatory gene is a repressor and the mutation is a recessive mutation. If the transcription level of the reporter gene is the same, the regulatory gene is an activator and the mutation is a dominant mutation 1. Translational fusion -- protein translocation and secretion Translational fusion means to put the reporter gene sequence downstream of yfg in the same open reading frame, thus constructing a fusion protein. The translation level of the reporter gene is the same as yfg 2. In this example, the lacZ gene is fused to the C-terminal of yfg. If the signal sequence is intact, the translated fusion protein will be directed to the Sec translocon when the inducer is present. However, the LacZ region will fold before the fusion protein is translocated, so the fusion protein will cause a "lethal jamming" of the sec translocon and lead to subsequent cell death. This is called the "inducer sensitive" phenotype 1. If the signal sequence is mutated, the fusion protein cannot be directed to the sec translocon, so it will not jam the translocon in the existence of inducer – the "inducer resistance" phenotype1. This set of experiments demonstrated the existence of signal peptide. References: 1. Shuman, H. a & Silhavy, T. J. The art and design of genetic screens: Escherichia coli.http://www.nature.com/nrmicro/journal/v10/n12/full/nrmicro2907.html Nat. Rev. Genet. 4, 419–31 (2003). 2. http://en.wikipedia.org/wiki/Lac_operon